Project Summary/Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most common cancer of the pancreas and 5-year survival rate for PDAC patients is a dreadful ~6%. PDAC genome sustains frequent deletion of tumor suppressor gene loci, most notably SMAD4, which is homozygously deleted in approximately 30% of cases. As loss of neighboring housekeeping genes can confer collateral lethality, I sought to determine whether loss of the metabolic gene malic enzyme (ME) 2 in the SMAD4 locus would create a cancer-specific metabolic vulnerability upon targeting its paralogous isoform ME3. Using in vitro and in vivo model system, I demonstrated that ME3 depletion in ME2 null PDAC cells causes cell death. Mechanistically, integrated metabolomic and molecular investigation of mitochondrial ME-deficient cells revealed diminished NADPH production and consequent high ROS which activates AMP activated protein kinase (AMPK), and which in turn suppresses BCAT2 (Branched chain amino acid transaminase 2) gene via sterol regulatory element-binding protein 1 (SREBP1)-directed transcription. BCAT2 catalyzes the transfer of the amino group from branched chain amino acids (BCAA) to alpha-ketoglutarate (?KG) thereby regenerating glutamate, which functions in part to support de novo nucleobase synthesis. In summary, this data shows that mitochondrial ME is required for pancreatic cancer survival and can be exploited as vulnerability for treatment. The goal of the project is to determine the druggability of ME in PDAC. In order to achieve this goal, I propose the following: 1) Systematic validation of Malic Enzyme as a therapeutic target in pancreatic cancer, 2) Mechanistic understanding of metabolic deregulation upon ME3 extinction; 3) Identification of selective ME3 inhibitors. By addressing the above aim, I will gain further insights into the structural features of ME3 and generate a blueprint for rational drug design to target ME3. Moreover, I will also identify the potential resistance or metabolic bypass mechanism of ME3 treatment by tracing metabolites such as BCAA and glutamine. Moreover, my unique understanding of the BCAA regulation will identify new avenues for targeting surrogate target of ME3. Finally, will undertake structure-function analysis of ME3 and validate the top hits identified by in-silico analysis.